CN114126617A

CN114126617A - Piezoelectric agonists for preventing or reversing abnormal amyloid deposition

Info

Publication number: CN114126617A
Application number: CN202080050355.5A
Authority: CN
Inventors: T·马尔姆; R·吉尼亚图林
Original assignee: EASTERN FINLAND, University of
Current assignee: EASTERN FINLAND, University of
Priority date: 2019-07-08
Filing date: 2020-07-07
Publication date: 2022-03-01
Also published as: EP3996702A1; US20220265646A1; JP2022540194A; WO2021005268A1

Abstract

The present invention relates to diagnosing, preventing, delaying or reversing the progression of pathologies associated with abnormal amyloid deposits such as for example Alzheimer's Disease (AD). More specifically, the method comprises the administration of specific molecules acting as piezoelectric agonists, such as Yoda1, Jedi1, Jedi2 or functional analogues thereof, capable of modulating the activation of microglia cells to an anti-inflammatory state and/or interfering with the formation of amyloidogenic peptides and/or increasing their efflux from the central nervous system. These agonists are useful in disease states associated with or at risk of cerebral amyloidosis, such as alzheimer's disease, parkinson's disease, stroke, head trauma, cerebral amyloid angiopathy, spongiform encephalopathy and pruritus, all of which have evidence of aberrant pro-inflammatory microglial activation.

Description

Piezoelectric agonists for preventing or reversing abnormal amyloid deposition

Technical Field

The present invention relates to diagnosing, preventing, delaying or reversing the pathological progression associated with abnormal amyloid deposits, such as that exemplified by Alzheimer's Disease (AD). More specifically, the method comprises the administration of specific molecules acting as piezoelectric agonists, such as Yoda1, Jedi1, Jedi2 or functional analogues thereof, capable of modulating the activation of microglia cells to an anti-inflammatory state and/or interfering with the formation of amyloidogenic peptides and/or increasing their efflux from the central nervous system. These agonists are useful in or at risk for disease states associated with cerebral amyloidosis (such as Alzheimer's disease, Parkinson's disease, one or more head trauma, stroke, cerebral amyloid angiopathy, spongiform encephalopathy and pruritus), all of which have evidence of aberrant pro-inflammatory microglial activation.

Technical Field

Microglia are highly dynamic cells that chemically and mechanically interact with their environment. Sensitive to their environmental changes in the human brain, they are balanced between pro-inflammatory and anti-inflammatory phenotypes (Hammond, Robinton and Stevens, 2018; Malm, Jay and Landreth 2015). Remodeling of microglia into a beneficial phenotype represents one of the promising strategies for improving brain function in neurodegenerative diseases such as Alzheimer's Disease (AD). One of the typical features is the accumulation of microglia around β amyloid or amyloid (a β) plaques (Malm et al, 2015). The functional state of these immune cells around the plaque, in particular their motility, can be a key determinant of the pathological processes in AD. Although the effects of chemical signaling on microglial function have been widely studied, the current understanding of mechanical signaling is very limited. Particularly due to the ability of neurodegenerative disorders to alter the mechanical properties of the brain, such as stiffness, which are known to decrease in the human brain with the progression of AD (ElSheikh et al, 2017; Murphy et al, 2016). Amyloid- β plaque formation is increased as a local stiffness due to its fibrous nature (-3 x 109 pa) compared to normal brain tissue stiffness (about 200-. As active as microglia, this cell type responds strongly to mechanical changes in its surroundings and is described as changing its morphology on a harder substrate and increasing fluidity towards a hardness gradient (Bollmann et al 2015; Moshayedi et al 2014). Similarly, for astrocytes, microglia upregulate inflammatory mediators and increase their inflammatory response when attracted to regions of increased stiffness (Bollmann et al 2015; Moshayedi et al 2014). This important function of microglia along heterogeneous extracellular environments, in connection with cellular remodeling, should be dependent on or ultimately lead to the activation of mechanosensitive channels. These newly discovered mechanosensitive piezoelectric receptors have rapidly attracted interest and have been shown to model astrocyte response to mechanical stimulation of extracellular Α β plaques (Velasco-Estevez et al, 2018). However, to date, the expression and functional role of mechanosensitive channels in brain-resident microglia has not been studied.

Mechanically Sensitive (MS) ion channels are molecular force sensors that are specialized for the rapid conversion of various mechanical forces into electrochemical signals for controlling critical biological activities such as touch, hearing, and blood pressure regulation. Therefore, it must be understood how this conversion process, known as mechanical gating, occurs precisely. Although significant progress has been made in the study of prokaryotic MS channels (i.e., MscL), the mechanical gating mechanisms of mammalian MS cation channels are relatively poorly understood.

Mechanosensitive piezoelectric receptors have recently become the most specific mechanosensitive sensors discovered since history (Coste et al, 2010; Coste et al, 2012). Mechanosensitive cell types are found not only in specialized organs such as the auditory or vestibular system, but also in other deformable excitable and non-excitable tissues of the whole body (Coste et al, 2010). The evolutionarily conserved family of piezoelectric proteins, including piezoelectric 1 and piezoelectric 2, have been identified as long sought for mammalian MS cation channels. The piezoelectric 2 receptor (piezoelectric 2R) subtype is predominantly expressed in the nociceptive system (Bron et al, 2014; Eijkelkamp et al, 2013; Kim et al, 2012), while the piezoelectric 1 channel is present in both the periphery and in different brain regions (Velasco-Estevez et al, 2018; Wu et al, 2017). In parallel with this study, Philip a. gottlieb laboratory found that soluble a β blocked the piezoelectric 1 receptor (piezoelectric 1R) (Maneshi et al, 2018), suggesting a close association between AD pathology and the functional state of brain cells expressing these mechanosensitive channels.

In mice, piezoelectric elements have been shown to play a key role in a variety of mechanical conduction processes, including tactile, auditory, and the sensation of shear stress associated with blood flow. In humans, mutations in piezoelectric genes that result in altered channel function are associated with many genetic diseases involving mechanical transduction. These studies demonstrate the functional importance and potential of piezoelectric channels as therapeutic targets. Piezoelectric channels represent a prototype of a mammalian mechanically susceptible cation channel. However, the mechanical gating mechanism is still unclear.

Chemical agonist Yoda1 at Ca²⁺Specifically activating (and modulating) piezoelectric 1 channels in an imaging assay provides a simple method for uniform stimulation of a large population of channels. The precise mechanism by which Yoda1 activates piezo1 is still unknown; the open state stabilized by Yoda1 had the same single channel conductance as the tension-gated open state, indicating that the two pore open conformations were similar. There are several reports indicating Yoda1 in various bodily functions such as bloodBeneficial effects in tube relaxation (Li et al, 2014; Wang et al, 2016), activation of the neuroprotective Ark pathway (dela Paz and Frangos 2018). In addition, Yoda 1-induced stimulation of endothelial release of ATP (Wang et al, 2016) may help indirectly activate these brain-resident immune cells, which are highly sensitive to the release of extracellular ATP.

WO2018232735a1 discloses a novel piezoelectric 1 chemical activator, called Jedi, which directly binds and activates the piezoelectric 1 (by adjusting its mechanical sensitivity). EP3006055 discloses a neurological implant comprising a biomaterial having an outer surface with random nano roughness (nanorough), and the use of said random nano roughness in the diagnosis and/or treatment of neurological disorders, and in the context of mammalian mechanical sensing ion channels for the destruction or prevention of glial scars. Blumenthal et al (2014) teach that the inhibition of mechanically induced cation channels whose distribution is altered by nanotopography, including piezo-1, eliminates the effects imposed by nanotopography and neuronal to astrocytic association.

AD (the most common form of senile dementia) will become more prevalent by the middle of this century, becoming a major global health problem with enormous impact on both individuals and society. While scientific breakthroughs over the past decades have expanded our understanding of the cellular and molecular basis of AD, there remains a lack of effective therapies to arrest disease progression and a focused effort is required to address this public health challenge. Stroke is the third leading cause of death and disability in industrialized countries, as well as the lack of effective therapeutic strategies. These diseases pose a significant socio-economic burden not only to the affected persons, but also to their relatives and caregivers. Pathological brain diseases such as AD, stroke, parkinson's disease, for example, manifest as abnormal microglial activation and abnormal protein accumulation in the extracellular space or intracellularly, as primary (AD, parkinson's disease) or secondary (stroke, head trauma) pathology.

Summary of The Invention

This is the first demonstration of the expression of piezoelectric receptors in microglia and their emerging role in regulating important functions of microglia, such as motility, phagocytosis and cytokine release. To date, evidence of microglia as one of the core roles in the development and progression of AD is a point of controversy. On the one hand, microglia are involved in AD pathogenesis by releasing inflammatory mediators (cytokines, chemokines, free radicals) that are known to contribute to the accumulation of beta-amyloid (Α β) and promote the death of nearby neurons in association with inflammation (Cai, Hussain and Yan, 2014; Parkhurst et al, 2013). On the other hand, microglia are also known to play a beneficial role in the production of amyloid plaques and the stimulatory clearance of anti-a β antibodies (Perlmutter et al, 1992). Thus, as the question of "chicken or egg" reappears, both a β plaque accumulation and clearance of AD microglia are involved.

The present inventors hypothesized that the presence of piezoelectric 1R channels in microglia cells that play a central role in AD-associated neuroinflammation could be targeted to clear Α β plaques in the brain. The failure of microglia to clean the brain in the early stages of AD onset was found to be due to a β inhibiting its function by piezoelectric 1R, consistent with Maneshi et al view in 2018. Current results indicate that microglia from both mouse and human samples express piezoelectric 1R channels and that their activation by Yoda1 greatly reduces the size of Α β plaques.

In summary, the present invention identifies specific molecules that are unexpectedly capable of interfering with the formation of amyloidogenic a β peptides and/or increasing their efflux from the central nervous system, and thus of reducing the burden of a β deposition in the brain in disease states associated with or at risk of cerebral amyloidosis (such as AD, cerebral amyloid angiopathy, spongiform encephalopathy and pruritus). In addition, the present invention identifies such molecules that modulate microglia activation status such that they have lower pro-inflammatory and greater phagocytosis, thereby providing protection in diseases where aberrant microglia activation persists. Furthermore, the invention relates to the use of the above compounds. The invention also relates to the use of the composition of the molecule in combination with other pharmacologically active substances capable of affecting the amyloid burden in the brain, such as antioxidants, anti-inflammatory agents, protease inhibitors, acetylcholinesterase inhibitors and the like.

The present disclosure is directed to piezoelectric agonists for treating neurodegenerative and/or neuroinflammatory diseases, or disorders or conditions associated with neurodegenerative and/or neuroinflammatory diseases. The neurodegenerative and neuroinflammatory diseases are evidenced by abnormal pro-inflammatory microglial activation.

The present disclosure is directed to piezoelectric agonists for the modulation of microglial function.

An object of the present disclosure is a method of treating a neurodegenerative and/or neuroinflammatory disease, or a disorder or condition associated with a neurodegenerative and/or neuroinflammatory disease, comprising administering to a subject in need thereof a piezoelectric agonist, wherein the agonist is for activating piezoelectric.

Another object of the invention is a method of treating a condition or disorder, such as a neurodegenerative disease or the like, or a condition or disorder associated with said neurodegenerative disease, the method comprising administering a piezoelectric agonist to a subject in need thereof, wherein the agonist is for activating piezoelectric. Another object of the invention is a method of treating a disease state associated with or at risk of cerebral amyloidosis, comprising administering a piezoelectric agonist to a subject in need thereof.

Yet another further object of the present invention is a method for determining one or more risks associated with the development or presence of a neurodegenerative and/or neuroinflammatory disease, or a disorder or condition associated with said neurodegenerative and/or neuroinflammatory disease, in a human subject, comprising the steps of:

a. providing a test sample and a control sample;

b. measuring baseline fluorescence intensities of the test sample and the control sample;

c. adding a piezoelectric agonist to the test sample;

d. measuring the fluorescence intensity of the test sample and the control sample;

e. determining the difference in fluorescence intensity between the test sample and the control sample,

wherein a decrease in the fluorescence intensity of the test sample compared to the fluorescence intensity of the control sample in step e is indicative of a decrease in the activity of the piezoelectric receptor in the test sample and is indicative of the risk of development or presence of a neurodegenerative disease and/or neuroinflammatory disease or a disorder or condition associated with the neurodegenerative disease and/or neuroinflammatory disease in the human subject.

A further object of the invention is the use of a piezoelectric agonist for the treatment of at least one disorder selected from the list consisting of alzheimer's disease, parkinson's disease, stroke, one or more head wounds, cerebral amyloid angiopathy, spongiform encephalopathy, cerebral amyloid angiopathy and pruritus, wherein the agonist is used to activate the piezoelectric. The condition is evidenced by abnormal pro-inflammatory microglial activation.

A further object of the invention is a method of modulating microglia function in a cell comprising contacting the cell with a piezoelectric agonist.

Drawings

Fig. 1A to 1H show the expression levels of piezoelectric receptors and their functional role in microglia. The corresponding relative gene expression levels for piezo1 (fig. 1A) and piezo2 (fig. 1B) are shown as fold changes and normalized to trigeminal cell piezo expression, each sample being repeated for m-3 organisms in independent experiments n-4. Expression of piezo1 in human induced pluripotent stem cell (hlsc) -derived microglia (fig. 1C) and human SV40 microglia line (fig. 1D). hIPSC-derived microglia by priming Ca²⁺The response was in response to the piezoelectric 1-specific agonist Yoda1 (fig. 1E). Surprisingly, pre-incubation with a β blocked the Yoda 1-induced Ca²⁺Reaction, indicating that a β specifically binds and prevents proper function of the piezo1 (fig. 1E). The response by Yoda1 and blockade by a β and the piezoelectric 1 inhibitor gadolinium (GdCl) were quantified in hiaps-derived microglia (fig. 1F) and SV-40 cells (fig. 1G). During the 2 day follow-up, a β reduced the motility of microglia (fig. 1H). All data are expressed as mean +/-SEM, where<.05,**p<.01,*＊*p<.001As analyzed by t-test or two-way ANOVA followed by Bonferroni post-hoc tests.

Figure 2A shows that activation of piezoelectricity with Yoda1 reduces microglial secretion of pro-inflammatory cytokines. Yoda1(Y20) at a concentration of 20. mu.M prevented LPS-induced secretion of pro-inflammatory IL-6, IL-8, MCP1 and increased secretion of anti-inflammatory IL-10 (FIG. 2). Using hlsc-derived microglia, n-4/group, one-way ANOVA followed by Bonferroni post-hoc testing,. p < 0.01; p < 0.001.

Figure 2B shows that activation of the piezo with Yoda1 prevented microglial cell death as analyzed by the cytotoxicity assay. An exemplary curve of microglial uptake of cytotoxic dye was measured over a 72 hour period. Quantification of dye uptake at the 60 hour time point indicated that 2-20 μ M Yoda1(Y2-Y20) protected hiPSC-microglia from cell death. n is 3 experiments. Positive control 200 μ M MPP +.

Figure 3 shows that Yoda1 treatment resulted in an increased immune positive rate of Iba1 and a reduction in a β deposits. Fig. 3A shows quantification of Iba1 staining in cortex and hippocampus. Representative images depict representative examples of i) control, ii) Yoda1, and iii GdCl treated hippocampus. Figure 3B shows quantification of WO2 staining in cortex and hippocampus. i) Control, ii) Yoda1 and iii) representative images of WO2 staining in the hippocampus of Gdcl treated animals. Figure 3C i) control and ii) high magnification images of Yoda1 treated animals show how Iba1 positive microglia (green) are recruited around a β deposits (indicated in red). The Yoda1 treated mice (ii) showed an increase in the number of Iba1 positive cells surrounding a β deposits compared to vehicle treated control (i). All data are expressed as mean +/-SEM, where p 05, p 01, p 001, as analyzed by t-test or two-way ANOVA, followed by Bonferroni post-hoc tests.

Fig. 4 shows that piezo-electricity 1 is expressed in developing neurons and peaks 3 days after culture (fig. 4A). The growth process of the neuronal processes was measured by using live incubation imaging. Figure 4B shows a typical example of vehicle and Yoda1 treated neurons at plating time (0h) and 6 days in vitro (DIV 6). Quantification of incucyte images taken over a period of 150 hours showed that vehicle-treated cells formed neurites as expected when plated at the same density, however, Yoda 1-treated cells showed more branching points (fig. 4C) and extended neurite length (fig. 4D) compared to vehicle-treated cells. Quantification of branch point (C) and neurite length (D). VEH-vehicle; yoda ═ Yoda 1. All data are expressed as mean +/-SEM, where p < 05, p < 001, as analyzed by t-test or two-way ANOVA, followed by Bonferroni post-hoc tests.

Fig. 5A to 5E show that Yoda1 prevents hypoxia-induced apoptosis. Yoda1 treatment was able to almost completely block hypoxia-induced early (fig. 5A) and late (fig. 5B) apoptosis of N2A cell model neurons. Veh-vehicle hypoxia and Yoda h-Yoda. Yoda1 was administered immediately after the stroke, once daily for three consecutive days thereafter. Lesion size was measured by MRI at 1DPI and 3 DPI. Yoda1 treated mice showed significantly smaller lesion sizes at both time points (fig. 5C). Representative MRI images from vehicle (top panel) and Yoda1 (bottom panel) treated mice, respectively (fig. 5D). Lesions appeared white. The ability of Yoda 1-treated ischemic mice to feel sticky plaques after stroke did not show a significant deficit (fig. 5E). All data are expressed as mean +/-SEM, where p 05, p 01, p 001, as analyzed by t-test or two-way ANOVA, followed by Bonferroni post-hoc tests.

Fig. 6A-6E show test results for Red Blood Cells (RBCs) obtained from 5XFAD mice early in AD pathology. In fig. 6: time point 1, mice 13-15w (FIG. 6A), time point 2, mice 15-17w (FIG. 6B), time point 3, mice 17-19w (FIG. 6C), time point 4, mice 19-21w (FIG. 6D) and time point five, mice 21-23w (FIG. 6E).

Detailed description of the inventionthe Alzheimer's Disease (AD) is characterized by a loss of neuronal function in the Central Nervous System (CNS). This loss of function occurs mainly around senile plaques, which are mainly composed of amyloid β deposits. The exact mechanism of neuronal death and loss of function is currently unclear.

The focus of current therapies is to dissolve amyloid beta deposits, and this approach has not been very successful. One approach is to use cholera toxin-B covalently linked to myelin basic protein (myelin basic protein). Therapies based on anti-amyloid β plaque antibodies (e.g., Bapineuzumab) developed by Johnson & Johnson in combination with Pfizer failed phase III clinical trials. In 2014, another β amyloid plaque targeting antibody developed by Roche/Genentech (crenezumab) failed to reach its phase II target. Thus, there is a real need to develop new targets to prevent or reverse neuronal loss of function caused by alzheimer's disease.

Stroke (the loss of brain function due to disturbed blood flow to the brain) places a tremendous burden on the affected individual's life and economy. The number of stroke patients is estimated to be 1500 ten thousand per year. Every year 500 million people die of stroke, and another 500 million patients have lifelong disabilities. The incidence of stroke is expected to continue to rise in the future in view of the aging population. Ischemic stroke accounts for more than 80% of all strokes and occurs when a cerebral artery is occluded, usually by thromboembolism. Brain cells lack oxygen and nutrients and begin to die within minutes after onset. Neuronal damage and death results in paralysis, loss of speech, vision, memory or coordination, and in the most severe cases, death. Despite extensive research, the only clinically available treatment for stroke is thrombolysis, in which the time window of treatment is only a few hours from the onset of symptoms. More effective treatments are urgently needed. The therapeutic effect of most conventional drugs is intended to arrest the acute phase of a stroke, which begins with the onset of restricted tissue blood flow and lasts for several hours. In fact, many treatments lose effectiveness if the medication is administered several hours later. This results in a lack of a realistic, effective time window for treating the patient. In the present invention, stroke protection is mediated by microglia.

Parkinson's disease is a long-term degenerative disorder of the central nervous system that primarily affects the motor system. Several mechanisms of brain cell loss are hypothesized. One mechanism consists of abnormal accumulation of the ubiquitin-binding protein α -synuclein in damaged cells. Other cell death mechanisms include dysfunction of the proteasome and lysosomal systems and decreased mitochondrial activity.

Head trauma may be as mild as a lump, bruise (contusion) or cut in the head, or may be moderate to severe in nature due to concussion, deep or open wound, one or more skull fractures or internal bleeding and brain damage. Head trauma is a broad term describing a series of injuries that occur to the scalp, skull, brain, and underlying tissues and blood vessels of the head. Depending on the extent of head trauma, head injury is also commonly referred to as brain injury or Traumatic Brain Injury (TBI).

Cerebral Amyloid Angiopathy (CAA), also known as congo red angiopathy, is a form of angiopathy in which amyloid deposits form in the walls of the central nervous system blood vessels. Amyloid is only present in the brain, and thus the disease is not associated with other forms of amyloidosis. CAA is defined as the deposition of a β in the pia mater and cerebral vessel walls. The cause of the increased a β deposition in sporadic CAA is still unclear and both increased peptide production and abnormal clearance are considered as potential causes.

Transmissible Spongiform Encephalopathies (TSEs) are a group of progressive, inevitable, fatal disorders associated with prions and affecting the brain (encephalopathy) and nervous system of many animals, including humans, cattle and sheep. According to the most extensive hypothesis, they are transmitted by prions, although other data suggest that they are associated with spiroplasma infections. Human TSEs include Creutzfeldt-Jack disease (Creutzfeldt-Jakob disease) -which has four major forms, sporadic (sCJD), hereditary/familial (fCJD), Iatrogenic (iCJD), and variant (vCJD) -Gerstmann-Straussler-Scheck syndrome (cJD)

syndrome), fatal familial insomnia, kuru and recently discovered variable protease-sensitive prion diseases. These disorders form a range of diseases where signs and symptoms overlap. TSE of non-human mammals including sheep scratchy condition, bovine spongeEncephalopathy (BSE), commonly known as mad cow disease, and Chronic Wasting Disease (CWD) of deer and elk, also known as zombie deer disease.

An active agent such as an antibody, nucleotide, small molecule or activated binding compound (for ion channel function) that binds piezoelectrically may be administered in the form of a pharmaceutical composition.

Preferably, the active agent is a piezoelectric agonist. More preferably, the agonist is Yoda1 having formula I

Jedi1 having formula II

Or Jedi2 having formula III

Or one or more functional analogs thereof. As used herein, the term "functional analog" refers to a compound having similar physical, chemical, biochemical, or pharmacological properties. The term "agonist" refers to substances that positively influence a process, e.g., substances that activate or stimulate a process, including chemical, biochemical, cellular, or physiological processes. It is understood that it includes, but is not limited to, these substances. "piezoelectric agonist" is understood to mean a molecule capable of activating a piezoelectric receptor. Piezoelectric agonists can bind to piezoelectric receptors. Examples of piezoelectric agonists are Yoda1, Jedi1, and Jedi2 and functional analogs thereof. Most preferably the agonist is Yoda 1. The amount of agonist used to activate the piezo is between 1nM and 50. mu.M. Preferably, the amount may be 100nM, 200nM, 300nM, 400nM, 500nM, 600nM, 700nM, 800nM, 900nM, 100 nM-1. mu.M, 500 nM-1. mu.M, 1. mu.M-25. mu.M, 1. mu.M-20. mu.M, 1. mu.M-15. mu.M, 1. mu.M-10. mu.M, 1. mu.M-5. mu.M, 5. mu.M-10. mu.M, 5. mu.M-15. mu.M, 10. mu.M-20. mu.M, 10. mu.M-25. mu.M, 10. mu.M-30. mu.M, 15. mu.M-30. mu.M, 20. mu.M-30. mu.M, 25. mu.M-30. mu.M, 30. mu.M-40. mu.M, 30. mu.M-50. mu.M, 40. mu.M-50. mu.M, 45. mu.M-50. mu.M, 1. mu.M, 2. mu.M, 3. mu.M, 4. mu.M, 5. mu.M, 6. mu.M, 7. mu.M, 8. mu.M, 9. mu.M, 10. mu.M, 11. mu.M, 12. mu.M, 13. mu.M, 14. mu.M, 15. mu.M, 16. mu.M, 17. mu.M, 18. mu.M, 19. mu.M, 20. mu.M, 21. mu.M, 22. mu.M, 23. mu.M, 24. mu.M, 25. mu.M, 26. mu.M, 27. mu.M, 28. mu.M, 29. mu.M, 30. mu.M, 40. mu.M or 50. mu.M. More preferably, the amount is 10. mu.M-50. mu.M.

An "effective amount" is an amount of one or more compounds or pharmaceutical compositions described herein that induces ion channel expression and/or abundance or induces ion channel activity. The amount alleviates a symptom found for the neurodegenerative and/or neuroinflammatory disease or a condition or disorder associated with said neurodegenerative and/or neuroinflammatory disease. Alleviation means, for example, preventing, treating, alleviating the symptoms of, or curing a disease (such as AD, parkinson's disease, stroke, one or more head wounds, cerebral amyloid angiopathy, spongiform encephalopathy, and pruritus) or disorder (e.g., plaque formation).

In one embodiment, a therapeutically effective dose of a compound disclosed herein, such as a piezoelectric agonist, is from about 0.1mg to about 2,000mg per day. The pharmaceutical composition should provide a dose of the compound of about 0.1mg to about 2,000 mg. In particular embodiments, the pharmaceutical dosage unit forms are prepared to provide from about 1mg to about 2,000mg, from about 10mg to about 1,000mg, from about 20mg to about 500mg, or from about 25mg to about 250mg of the active ingredient or combination of essential ingredients per dosage unit form. In particular embodiments, the pharmaceutical dosage unit form is prepared to provide about 10mg, 20mg, 25mg, 50mg, 100mg, 250mg, 500mg, 1000mg or 2000mg of the active ingredient.

One object of the present invention is a piezoelectric agonist for use in the treatment of a neurodegenerative and/or neuroinflammatory disease, or a condition or disorder associated with a neurodegenerative and/or neuroinflammatory disease.

One aspect of the invention is a piezoelectric agonist for use in the prevention of a neurodegenerative and/or neuroinflammatory disease, or a condition or disorder associated with a neurodegenerative and/or neuroinflammatory disease.

In embodiments, the disease or disorder or condition to be treated and/or prevented is an inflammatory disease or disorder or condition, preferably a neuroinflammatory disease or disorder or condition.

In an embodiment, the piezoelectric agonist is for use in therapy, wherein the piezoelectric is piezoelectric 1 or piezoelectric 2. In embodiments, the piezoelectric agonist is for use in therapy, wherein the piezoelectric is from a mouse or a human. In an embodiment, the piezoelectric agonist is for use in therapy, wherein the piezoelectric agonist is for activating piezoelectricity.

In an embodiment, the piezoelectric agonist is for use in therapy, wherein the piezoelectric agonist is selected from the group consisting of Yoda1, Jedi1, Jedi2, and functional analogs thereof. In a preferred embodiment for use in therapy, the piezoelectric agonist is Yoda 1.

One object of the present invention is a piezoelectric agonist for the modulation of microglia function. In embodiments, the piezoelectric agonist is used in therapy, wherein the microglia function to be modulated is motility, phagocytosis and/or cytokine release.

According to one embodiment, the modulated microglial function is phagocytosis and/or cytokine release. Activation of the PiezoRs1 resulted in a decrease in microglial factor production and enhanced microglial phagocytosis. Microglia-associated cytokines are, for example, pro-inflammatory cytokines such as IL-1 β, IFN γ, IL-6, IL-2, and TNF α, but various other cytokines may also be included. Representative cytokines include, but are not limited to, the group consisting of: interleukin-1 a (IL-1a), interleukin-3 (IL-3), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8/CXCL8), interleukin-10 (IL-10), interleukin-12 (IL-12), interleukin-13 (IL-13), interleukin-15 (IL-15), interleukin-17 (IL-17), interleukin-18 (IL-18), tumor necrosis factor alpha (TNF-alpha), Interferon beta (INF-beta), interferon alpha (INF-alpha), interferon gamma (INF-gamma), granulocyte monocyte colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), monocyte chemotactic protein-1 (MCP-1/CCL2), macrophage inflammatory protein-1 alpha (MIP-1 alpha/CCL 3), macrophage inflammatory protein-13 (MIP-13/CCL4), RANTES (CCL5), Eotaxin (Eotaxin) (CCL 11), Variable Endothelial Growth Factor (VEGF), Endothelial Growth Factor (EGF) and Fibroblast Growth Factor (FGF).

In embodiments, the piezoelectric agonist is used in a treatment in which amyloid beta accumulation is inhibited.

In an embodiment, the piezoelectric agonist is for use in a treatment in which amyloid beta plaque burden is reduced.

In embodiments, the piezoelectric agonist is used in a therapy wherein the piezoelectric agonist is used in combination with at least one molecule selected from the group consisting of: arachidonoyl ethanolamide, 2-arachidonoyl glycerol, palmitoyl ethanolamide, oleoyl ethanolamide, and linoleoyl ethanolamide.

In an embodiment, the piezoelectric agonist is for use in the treatment, wherein the neurodegenerative and/or neuroinflammatory disease, or a condition or disorder associated with a neurodegenerative and/or neuroinflammatory disease is selected from the group consisting of: alzheimer's disease, stroke, Parkinson's disease, one or more head wounds, cerebral amyloid angiopathy, spongiform encephalopathy, cerebral amyloid diseases and pruritus. The neurodegenerative and/or neuroinflammatory disease is manifested by abnormal pro-inflammatory microglial activation.

In embodiments, the piezoelectric agonist is used in a pharmaceutical composition for prophylactic or therapeutic treatment of a neurodegenerative and/or neuroinflammatory disease, or a condition or disorder associated with a neurodegenerative and/or neuroinflammatory disease.

One object of the present invention is a piezoelectric agonist for use in the treatment of a neurodegenerative disease or a condition or disorder associated with said neurodegenerative disease. More preferably, the neurodegenerative disease or the condition or disorder associated with said neurodegenerative disease comprises at least one of: alzheimer's disease, Parkinson's disease, stroke, one or more head wounds, cerebral amyloid angiopathy, spongiform encephalopathy and pruritus. The neurodegenerative disease or disorder or condition is manifested by abnormal pro-inflammatory microglial activation.

Preferably, the patient is a human. Treatment is meant to include, for example, preventing, treating, alleviating the symptoms of, or curing a disease or disorder (i.e., a neurodegenerative disease or disorder or condition associated with such a neurodegenerative disease that is manifested by aberrant proinflammatory microglial activation).

Preferred is a method according to the invention, wherein said neurodegenerative disease or a condition or disease or disorder associated with said neurodegenerative disease is a cerebral amyloidosis disease and is selected from the group consisting of alzheimer's disease, parkinson's disease, stroke, one or more head trauma, cerebral amyloid angiopathy, spongiform encephalopathy and pruritus.

The invention also includes methods for treating a subject at risk of suffering from a neurodegenerative disease, wherein a therapeutically effective amount of the above-described modulators is provided. The risk of developing a disease can be caused by, for example, the family history of the disease, the genotype predisposed to the disease, or the phenotypic symptoms predisposed to the disease. A further aspect of the invention is the use of a modulator of the expression and/or biological activity of an ion channel for the preparation of a pharmaceutical composition for the treatment or prevention of a neurodegenerative disease or a condition or disorder associated with said neurodegenerative disease. Preferably, the modulator is an activator of the expression and/or biological activity of an ion channel as described herein.

The invention also provides pharmaceutical compositions comprising modulators for activating or inhibiting piezoelectricity, such as the piezoelectric activators Yoda1, Jedi1, or Jedi2, or functional analogs thereof. According to specific examples of the present invention, the pharmaceutical composition may further comprise pharmaceutically acceptable excipients, carriers, adjuvants, solvents, and combinations thereof.

The present invention provides methods of treating, preventing or ameliorating a disease or disorder comprising administering a safe and effective amount of a pharmaceutical combination comprising a compound and one or more therapeutically active agents. Wherein the pharmaceutical combination comprises one or more additional drugs for the treatment of a piezoelectric related disorder.

It will also be appreciated that certain compounds of the invention may be present in free form for use in therapy or, where appropriate, as pharmaceutically acceptable derivatives or prodrugs thereof. Pharmaceutically acceptable derivatives include pharmaceutically acceptable salts, esters, salts of such esters, or any other adduct or derivative that, when administered to a patient in need thereof, is capable of directly or indirectly providing a compound or metabolite or residue thereof as otherwise described herein.

In addition to the compounds of the present invention, the pharmaceutical compositions of the present invention also comprise one or more other active ingredients. As used herein, "pharmaceutically acceptable excipient" means a pharmaceutically acceptable material, composition or vehicle that participates in imparting a form or consistency (consistency) to a pharmaceutical composition. Suitable pharmaceutically acceptable excipients will vary depending on the particular dosage form selected. In addition, suitable pharmaceutically acceptable excipients may be selected for their specific function in the composition. Suitable pharmaceutically acceptable excipients include the following types of excipients: diluents, fillers, binders, disintegrants, lubricants, glidants, granulating agents, coating agents, wetting agents, solvents, solubilizing agents, suspending agents, emulsifiers, sweeteners, flavoring agents, flavor masking agents, colorants, anti-caking agents, wetting agents, chelating agents, plasticizers, viscosity increasing agents, antioxidants, preservatives, stabilizers, surfactants, and buffers. One skilled in the art will appreciate that certain pharmaceutically acceptable excipients may serve more than one function, and may serve alternate functions, depending on how many excipients are present in the formulation and which other ingredients are present in the formulation.

Pharmaceutical compositions comprise a compound disclosed herein and a pharmaceutically acceptable excipient, carrier, adjuvant, vehicle, or combination thereof, the method comprising admixing the various ingredients.

The target molecules of the present invention may be used alone or in combination with other selected therapeutic agents for the preparation of pharmaceutical compositions for specific therapeutic purposes. The selected therapeutic agents used in conjunction with the target molecules of the present invention may be selected from: antioxidants, protease inhibitors, acetylcholinesterase inhibitors, anticonvulsants, antipsychotics, atypical antipsychotics, antidepressants, dopamine agonists, GABA agonists, memory-improving drugs, anti-inflammatory drugs, analgesics (e.g., opioids, salycilates, pyrazoles, indoles, anthranilic acids, arylpropionic acids, arylacetic acids, oxicams, pyranocarboxylic acids, glucocorticoids, cox2 inhibitors, and acetaminophen).

The compounds of the present invention will generally be formulated in a dosage form suitable for administration to a patient by the desired route of administration. For example, dosage forms include those suitable for oral administration, such as tablets, capsules, wafers (caplets), pills, lozenges, powders, syrups, elixirs, suspensions, solutions, emulsions, sachets and cachets; those suitable for parenteral administration, such as sterile solutions, suspensions, and powders for reconstituted transdermal administration, such as transdermal patches; those suitable for rectal administration, such as suppository inhalants, such as aerosols, solutions and dry powders, and those suitable for topical administration, such as creams, ointments, lotions, solutions, pastes, sprays, foams and gels.

The pharmaceutical compositions provided herein can be administered parenterally by injection, infusion, or implantation for local or systemic administration. As used herein, parenteral administration includes intravenous, intraarterial, intraperitoneal, intrathecal, intracerebroventricular, intraurethral, intrasternal, intracranial, intramuscular, intrasynovial and subcutaneous administration. In addition, administration may be oral or intravenous.

Pharmaceutical compositions intended for parenteral administration may comprise one or more pharmaceutically acceptable carriers and excipients, including but not limited to aqueous vehicles, water-miscible vehicles, non-aqueous vehicles, antimicrobial or preservative agents to prevent microbial growth, stabilizers, solubility enhancers, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, wetting or emulsifying agents, complexing agents, chelating agents (sequestrant) or chelating agents (chelating agents), cryoprotectants, lyoprotectants, thickening agents, pH adjusting agents, and inert gases.

The pharmaceutical compositions provided herein can be formulated for single or multiple dose administration. The single dose formulations are packaged in ampoules, vials or syringes. Multi-dose parenteral formulations must contain bacteriostatic or fungistatic concentrations of antimicrobial agents. As is known and practiced in the art, all parenteral formulations must be sterile.

In one embodiment, a compound of the invention or a pharmaceutical composition thereof may be administered once or according to a dosing regimen, wherein multiple doses are administered at different time intervals over a given period of time. For example, the dose may be administered once, twice, three times or four times daily. Typically, dosage levels of 0.0001 to 10mg/kg body weight are administered to the patient daily to achieve effective modulation of the piezo.

The compounds of the invention may be administered concurrently with one or more other therapeutic agents, or before or after the other therapeutic agents. The compounds of the invention may be administered alone by the same or different route of administration, or together in the same pharmaceutical composition as the other agent.

Piezoelectric channels are useful not only for reducing plaque in dementia states, but also as biomarkers of predisposing to pre-symptomatic alzheimer's disease. This approach is based on the large expression of piezoelectric channels in Red Blood Cells (RBCs) and also blood mononuclear cells. The latter may be reduced or rendered non-functional in the early stages of AD due to the known inhibitory effect of a β on piezo1 protein. A minimally invasive method of obtaining a small blood sample and then flow cytometry testing the function of the piezoelectric 1 channel activated by Yoda1 can be used to broadly screen for AD susceptibility in middle-aged subjects when it is most effective to prevent subsequent dementia.

One object of the present invention is a method for determining the risk associated with the development or presence of a neurodegenerative and/or neuroinflammatory disease or a disorder or condition associated with said neurodegenerative and/or neuroinflammatory disease in a human subject, comprising the steps of:

a. providing a test sample and a control sample;

b. measuring the baseline fluorescence intensity of the sample and the control sample;

c. adding a piezoelectric agonist to the test sample;

e. the average fluorescence intensity of the test sample is determined,

wherein a decrease in the fluorescence intensity of the test sample in step e compared to the fluorescence intensity of the control sample is indicative of a decrease in the activity of the piezoelectric receptor in the test sample and is indicative of the risk of developing or presenting a neurodegenerative and/or neuroinflammatory disease or a disorder or condition associated with the neurodegenerative and/or neuroinflammatory disease in the human subject.

One object of the present invention is a method for determining the risk associated with the development or presence of a neurodegenerative disease or a condition or disorder associated with said neurodegenerative disease in a human subject, comprising the steps of:

a. providing a test sample;

b. adding a calcium indicator to the sample;

c. measuring a baseline fluorescence intensity of the sample;

d. adding a piezoelectric agonist to the test sample;

e. measuring the fluorescence intensity of the test sample;

f. adding a calcium ionophore to said test sample;

g. measuring the fluorescence intensity of the test sample; and

h. the mean fluorescence intensity of the test sample is determined,

wherein a decrease in the fluorescence intensity of the test sample in step e as compared to the fluorescence intensity of step c is indicative of a decrease in the activity of the piezoelectric receptor in the test sample and is indicative of a risk of development or presence of a neurodegenerative disease or a condition or disorder associated with the neurodegenerative disease in the human subject.

One object of the present invention is a method of determining the risk associated with the development or presence of a neurodegenerative disease or a condition or disorder associated with said neurodegenerative disease in a human subject, comprising the steps of:

a. providing a test sample and a control sample;

b. adding a calcium indicator to the test sample and/or control sample;

c. measuring baseline fluorescence intensities of the test sample and the control sample;

d. adding a piezoelectric agonist to the test sample;

e. measuring the fluorescence intensity of the test sample and the control sample;

f. adding a calcium ionophore to the test sample and/or control sample;

g. measuring the fluorescence intensity of the test sample and the control sample; and

h. the average fluorescence intensity of the test sample is determined,

wherein a decrease in the fluorescence intensity of the test sample in step e as compared to the fluorescence intensity of the control sample is indicative of a decrease in the activity of the piezoelectric receptor in the test sample and is indicative of the risk of development or presence of a neurodegenerative disease or a condition or disorder associated with the neurodegenerative disease in the human subject. Calcium indicators, such as synthetic Ca²⁺The dyes Fluo-4, Fluo-5F, Fluo-4FF, Rhod-2, X-Rhod-5F, Oregon Green 488BAPTA-6F, Fluo-8, Fluo-8 high affinity, Fluo-8 low affinity, Oregon Green BAPTA-1, Cal-520, Rhod-4, Asante calcium Red and X-Rhod-1, and the genetically encoded Ca²⁺Indicators such as GCaMP 6-slow, medium, and fast variants can be used in the present invention. Preferably, the indicator is Fluo-4.

Calcium ionophores were used as positive controls in the test. Calcium ionophores release all Ca resources (i.e., Ca-uncouplers) and indicate that the cells are viable and functional. Calcium ionophores such as ionomycin, calcimycin (a23187) or calcium ionophore V (K23E1) may be used in the present invention. Preferably, the ionophore is ionomycin.

Detection of Ca in test samples compared to baseline measurements²⁺A reduction in influx of at least 10% indicates a decrease in piezoelectric receptor activity in the test sample and an increase in amyloid- β accumulation in the human subject. Preferably, Ca²⁺The inflow reduction may be 10-100%,10-90%, 10-85%, 10-80%, 10-75%, 10-70%, 10-65%, 10-60%, 10-55%, 10-50%, 10-45%, 10-40%, 10-35%, 10-30%, 10-25%, 20-100%, 20-90%, 20-85%, 20-80%, 20-75%, 20-70%, 20-65%, 20-60%, 20-55%, 20-50%, 20-45%, 20-40%, 20-35%, 30-100%, 30-90%, 30-85%, 30-80%, 30-75%, 30-70%, 30-65%, 30-60%, 30-55%, 30-50%, 30-45%, 40-100%, 40-90%, 40-85%, 40-80%, 40-75%, 40-65%, 40-60%, 50-100%, 50-90%, 50-85%, 50-80%, 50-75%, 50-70%, or 10-70%, 10-50%, 20-50%, 20-90%, 20-40-35%, 30-85%, 30-90%, 30-80%, 40-75%, 40-60%, 40-50-80%, or 40-80% of the like, 60-100%, 60-90%, 60-85%, 60-80%, 60-75%, 70-100%, 80-100%, or 90-100%.

The most preferred biomarkers according to the present invention are piezo1 and piezo2, or a combination thereof.

Generally, molecular biomarkers can be detected in bodily fluids such as blood and plasma, lymph, fluid, and/or urine. In a preferred embodiment, the molecular biomarkers can be detected in blood, in particular in erythrocytes or blood mononuclear cells. Biomarkers can be easily examined. For the purposes of the present invention, biomarker protein (piezoelectric) activity is reduced when a subject has a condition associated with amyloid β accumulation, or is at risk of developing or has a neurodegenerative disease and has an increased response to treatment with a piezoelectric agonist.

Natural cannabinoid receptor ligands, also known as "endocannabinoids", are commonly represented by arachidonoyl ethanolamide (anandamide), AEA) and 2-arachidonoyl glycerol (2 AG). Tissue levels of endocannabinoids are maintained by a balance between biosynthesis (e.g., phospholipase D and diacylglycerol lipase dependent and other pathways), cellular uptake and enzymatic degradation (primarily but not limited to Fatty Acid Amide Hydrolase (FAAH) and/or monoacylglycerol lipase (MAGL)). Since the discovery of CB₁And CB₂GPCRs such as GPR₁₈、GPR₅₅、GPR₁₁₉And TRP they are considered members of the cannabinoid family. (CB ═ cannabinoid; GPCR ═ G protein-coupled receptor and TRP ═ transient receptor potential).

The endocannabinoids (MAGL and FAAH) have been shown to have a strong anti-inflammatory effect. This is based on the accumulation of endocannabinoids such as 2-AG and AEA, limiting overuse and psychotropic (psychotropic), which may occur in case of exogenous administration of cannabinoids. Furthermore, such inhibition limits the production of downstream endogenous cannabinoids (endocanans), such as arachidonic acid and its derivatives, which exert a strong pro-inflammatory effect. A dual synergistic protective effect is possible with the combination therapy of endocannabinoids and piezo agonists.

The combined approach of the present inventors to treat plaque would benefit primarily from the combination of a piezoelectric agonist with a potent agent that accumulates the endocannabinoids 2-AG and AEA. The most efficient method for accumulating 2-AG and AEA is to block the enzymes MAGL and FAAH with the currently available super-strong compounds JJKK-048 and JZP-372A, respectively. This new approach is consistent with recent data showing that 2-AG plays a protective role in CNS injury models, regulates microglia entry into an anti-inflammatory state via the CB2 receptor, and reduces the expression of pro-inflammatory cytokines.

Embodiments of the invention may comprise one or more molecules selected from mammalian cannabinoids. Preferred embodiments of the present invention may be formulated to result in the presence or increased activity of at least one active ingredient comprising at least one molecule selected from the group consisting of: arachidonoyl Ethanolamide (AEA), 2-arachidonoyl glycerol (2AG), Palmitoyl Ethanolamide (PEA), Oleoyl Ethanolamide (OEA), and Linoleoyl Ethanolamide (LEA). PEA, OEA and LEA are N-acylethanolamides. One or more embodiments of the present invention may have at least one active ingredient selected from the group consisting of: URB597, URB937, AM374, ARN2508, BIA 10-2474, BMS-469908, CAY-10402, JNJ-245, JNJ-1661010, JNJ-28833155, JNJ-40413269, JNJ-42119779, JNJ-42165279, LY-2183240, cannabidiol, MK-3168, MK-4409, MM-433593, OL-92, OL-135, PF-622, PF-750, PF-3845, PF-04457845, PF-04862853, RN-450, SA-47, SA-73, SSR-411298, ST-4068, TK-25, URB524, URB597(KDS-4103), URB694, URB937, VER-156084, V-158866, AM3506, AM6701, CAY10435, CAY 99, 104FP, JJJJKK-048, JKK-048, JNJ-651, JNJ-9634, JNJNJNJNJ-369634, JNJNJNJ-A, KML29, JNJNJ-A, KML29, JNO-3645, JNO-2, JNO-III, JHB8, JNO-III, JNO-2, JNO-III, JNO-III-2, JNO-III, and their derivatives, and their formula, OL-135, OL92, PF-04457845, SA-57, ST4070, URB880, URB937, indomethacin, MK-886, resveratrol, cis-resveratrol, aspirin, COX-1 inhibitor II, loganin, tenidap, SC560, FR122047 hydrochloride, valerylsalicylic acid, FR122047 hydrate, ibuprofen, TFAP, 6-methoxy-2-naphthylacetic acid, meloxicam, APHS, etodolac, meloxicam sodium salt, N- (4-acetamidophenyl) indomethacin amide, N- (2-phenylethyl) indomethacin amide, N- (3-pyridyl) indomethacin amide, indomethacin heptyl ester, SC236, sulindac sulfide, pravastatin, naproxen sodium salt, meclofen sodium, ibuprofen, S-ibuprofen, piroxicam, ketoprofen, S-ketoprofen, R-ibuprofen, ebselen, ETYA, diclofenac diethylamine, flurbiprofen, fexofenadine, pterostilbene, pterocarpus marsupium (pterocarpus marsupium), 9, 12-octadecadienoic acid, ketorolac (tromethamine salt), NO-indomethacin, S-flurbiprofen, sedanolide, green tea extracts (e.g., epicatechin), linuron, lornoxicam, rac ibuprofen-d 3, ampirxicam, zaltoprofen, 7- (trifluoromethyl) 1H-indole-2, 3-dione, aceclofenac, acetyl salicylic acid-d 4, S-ibuprofen lysine salt, loxoprofen, CAY10589, ZU-6, isoicam, analgin, YS121, and MEG (mercaptoethylguanidine). Preferred embodiments may incorporate 2,3, 4, 5, 6 or even more cannabinol (cannabinolic) support compounds or enzymes.

Solvents for the purposes of the present invention may not interfere with the biological activity of the solute. Examples of suitable solvents include, but are not limited to, water, methanol, ethanol, oleic acid, and acetic acid, or organic solvents such as THF, DMF, Dichloromethane (DCM), ethyl acetate (EtOAc), or acetonitrile. Preferably, the solvent used is a pharmaceutically acceptable solvent. Examples of suitable pharmaceutically acceptable solvents include, but are not limited to, methylcellulose, water, ethanol, and acetic acid.

One aspect of the invention is a piezoelectric agonist for use in treating a neurodegenerative and/or neuroinflammatory disease, or a condition or disorder associated with a neurodegenerative and/or neuroinflammatory disease. In an embodiment, the piezoelectric agonist is for use in therapy, wherein the piezoelectric is piezoelectric 1 or piezoelectric 2. In embodiments, the piezoelectric agonist is for use in therapy, wherein the piezoelectric is from a mouse or a human.

In an embodiment, the piezoelectric agonist is for use in therapy, wherein the piezoelectric agonist is for modulating microglial function. In a preferred embodiment, the piezoelectric agonist is used in therapy, wherein the microglial function to be modulated is motility, phagocytosis and/or cytokine release.

In an embodiment, the piezoelectric agonist is for use in therapy, wherein the piezoelectric agonist is for activating piezoelectricity.

In an embodiment, the piezoelectric agonist is for use in therapy, wherein the piezoelectric agonist is used in combination with at least one molecule selected from the group consisting of: arachidonoyl ethanolamide, 2-arachidonoyl glycerol, palmitoyl ethanolamide, oleoyl ethanolamide, and linoleoyl ethanolamide.

In an embodiment, the piezoelectric agonist is for use in therapy, wherein the neurodegenerative and/or neuroinflammatory disease or disorder or condition associated with a neurodegenerative and/or neuroinflammatory disease is selected from alzheimer's disease, stroke, parkinson's disease, one or more head wounds, cerebral amyloid angiopathy, spongiform encephalopathy, cerebral amyloid angiopathy, and pruritis. The neurodegenerative or neuroinflammatory disease is manifested by abnormal pro-inflammatory microglial activation.

In embodiments, the piezoelectric agonist is used in a pharmaceutical composition for prophylactic or therapeutic treatment of a neurodegenerative and/or neuroinflammatory disease or a condition or disorder associated with a neurodegenerative and/or neuroinflammatory disease.

One aspect of the invention is a method of treating a neurodegenerative and/or neuroinflammatory disease or a disorder or condition associated with a neurodegenerative and/or neuroinflammatory disease, the method comprising administering to a subject in need thereof a piezoelectric agonist, wherein the agonist is for activating piezoelectric.

In an embodiment of the method, the piezoelectric agonist is selected from the group consisting of Yoda1, Jedi1, Jedi2, and functional analogs thereof. In a preferred embodiment of the method, the piezoelectric agonist is Yoda 1.

In an embodiment of the method, the piezoelectric agonist is used in combination with at least one molecule selected from the group consisting of: arachidonoyl ethanolamide, 2-arachidonoyl glycerol, palmitoyl ethanolamide, oleoyl ethanolamide, and linoleoyl ethanolamide.

The present invention is directed to piezoelectric agonists for the modulation of microglial function. In a more preferred embodiment, the piezoelectric body itself is either piezoelectric 1 or piezoelectric 2. In a more preferred embodiment, the piezoelectric is from a mouse or a human. According to a preferred embodiment, the agonist is used to activate the piezo. According to another preferred embodiment, the agonist is Yoda1, Jedi1, Jedi2 or a functional analogue thereof. According to a preferred embodiment, the microglial function to be modulated is motility, phagocytosis and/or cytokine release. In a preferred embodiment, the accumulation of beta-amyloid is inhibited. In yet another preferred embodiment, the load of β -amyloid plaques is reduced.

According to a preferred embodiment, the piezoelectric agonist is for use in the treatment of a disorder or condition associated with cerebral amyloidosis disease. According to yet a more preferred embodiment, when the piezoelectric agonist is for use in the treatment of a neurodegenerative disease or a condition or disorder associated with said neurodegenerative disease, the piezoelectric agonist is used in combination with at least one molecule selected from the group consisting of: arachidonoyl Ethanolamide (AEA), 2-arachidonoyl glycerol (2AG), Palmitoyl Ethanolamide (PEA), Oleoyl Ethanolamide (OEA), and Linoleoyl Ethanolamide (LEA). Piezoelectric agonists may also be used when the condition or disorder associated with a neurodegenerative disease is selected from alzheimer's disease, parkinson's disease, stroke, one or more head trauma, cerebral amyloid angiopathy, spongiform encephalopathy and pruritus. The neurodegenerative disease is manifested by abnormal pro-inflammatory microglial activation. According to a preferred embodiment, the piezoelectric agonist is for use in a pharmaceutical composition for the prophylactic or therapeutic treatment of a disorder or condition associated with cerebral amyloidosis disease.

According to one embodiment is a method of treating a condition or disorder associated with a neurodegenerative disease or the neurodegenerative disease itself, the method comprising administering to a subject in need thereof a piezoelectric agonist, wherein the agonist is for activating piezoelectric. According to a preferred embodiment is a method of treating a condition or disorder wherein a piezoelectric agonist is used in combination with at least one molecule selected from the group consisting of AEA, 2-AG, PEA, OEA and LEA.

A method of treating a neurodegenerative disease or a condition or disorder associated with said neurodegenerative and/or neuroinflammatory disease is also a preferred aspect of the invention, which comprises at least one of the following diseases: alzheimer's disease, Parkinson's disease, stroke, one or more head wounds, cerebral amyloid angiopathy, spongiform encephalopathy, cerebral amyloid diseases and pruritus.

A method for determining the risk associated with the development or presence of a neurodegenerative and/or neuroinflammatory disease or a condition or disorder associated with said neurodegenerative and/or neuroinflammatory disease in a human subject, comprising the steps of:

a. providing a test sample and a control sample;

c. adding a piezoelectric agonist to the test sample;

wherein a decrease in the fluorescence intensity of the test sample in step e compared to the fluorescence intensity of the control sample is indicative of a decrease in the activity of the piezoelectric receptor in said test sample and is indicative of the risk of development or presence of a neurodegenerative and/or neuroinflammatory disease or a disorder or condition associated with said neurodegenerative and/or neuroinflammatory disease in said human subject, which is also an aspect of the present invention.

A method for determining one or more risks associated with the development or presence of a neurodegenerative and/or neuroinflammatory disease or a condition or disorder associated with said neurodegenerative and/or neuroinflammatory disease in a human subject, comprising the steps of:

a. providing a test sample;

b. adding a calcium indicator to the sample;

c. measuring a baseline fluorescence intensity of the sample;

d. adding a piezoelectric agonist to the test sample;

e. measuring the fluorescence intensity of the test sample;

f. adding a calcium ionophore to said test sample;

g. measuring the fluorescence intensity of the test sample; and

h. the average fluorescence intensity of the test sample is determined,

wherein a decrease in the fluorescence intensity of the test sample in step e compared to the fluorescence intensity of step c is indicative of a decrease in the activity of the piezoelectric receptor in the test sample and is indicative of the presence or risk of a neurodegenerative disease or a condition associated with said neurodegenerative disease in said human subject, which is also an aspect of the present invention. According to a preferred embodiment of the method, the sample used in the method consists of red blood cells, blood mononuclear cells, serum, plasma or whole blood. More preferably, the sample consists of red blood cells or blood mononuclear cells.

In an embodiment of this method, the neurodegenerative and/or neuroinflammatory disease or the disorder or condition associated with said neurodegenerative and/or neuroinflammatory disease comprises at least one of: alzheimer's disease, Parkinson's disease, stroke, one or more head wounds, cerebral amyloid angiopathy, spongiform encephalopathy, cerebral amyloid diseases and pruritus. The neurodegenerative or neuroinflammatory disease is manifested by abnormal pro-inflammatory microglial activation.

In an embodiment of this method, the disorder, disease or condition is or is associated with at least one neurodegenerative and/or neuroinflammatory disease selected from: alzheimer's disease, Parkinson's disease, stroke, one or more head wounds, cerebral amyloid angiopathy, spongiform encephalopathy, cerebral amyloid diseases and pruritus.

The use of a piezoelectric agonist for the treatment of at least one condition selected from the group consisting of: alzheimer's disease, Parkinson's disease, stroke, one or more head wounds, cerebral amyloid angiopathy, spongiform encephalopathy, cerebral amyloid diseases and pruritus, wherein the agonist is used to activate piezoelectricity.

Also a further aspect of the invention is a method of modulating microglial function in a microglial cell comprising contacting the cell with a piezoelectric agonist.

Examples

Materials and methods

qRT-PCR

To demonstrate the expression levels of the piezo receptors (piezo 1R and piezo 2) in different microglia cell types, the inventors used qPCR (TaqMan kit, Tsher Scientific). The nucleic acid concentration was measured at 260 nm. The purity of the extracted RNA was determined as a ratio of 260nm/280nm, with expected values between 1.8 and 2. Next, the RNA samples were reverse transcribed into cDNA and primed single stranded RNA reactions were performed using random hexamer primers (ThermoFisher Scientific) according to the manufacturer's protocol. Two different primers were used with FAM fluorescent markers: mouse piezo-1 (P17113-005D08, ThermoFisher Scientific) and piezo 2(P17113-005D09, ThermoFisher Scientific) primers were used for BV2, mouse microglia and primary mouse trigeminal cultured cells, human piezo-1 (P17113-005D10, ThermoFisher Scientific) and piezo 2(P17113-005D11, ThermoFisher Scientific) were used for human postmortem cells, iPSC-derived human microglia and sv40 cell line (human immortalized microglia). PCR amplification of the cDNA was quantified using a StepOnePelus real-time PCR system (ThermoFisher Scientific). Each sample was repeated 3 times.

Calcium imaging

To investigate calcium influx induced by natural piezoelectric 1R expressed in microglia, the inventors used a short Yoda1 application (2s, 50 μ M if not otherwise stated). For this purpose, human IPSC-derived microglia and SV-40 (immortalized human microglia) cells were seeded on 7mm coverslips 1 day before the imaging experiment. Cells were loaded with calcium sensitive fluorescent dye Fluo-4AM (5. mu.M) for 30 min at 37 ℃ and then incubated for 15 min with a wash in Ab20pM, 50. mu.M GdCl and alkaline solution (BS) as a control at 37 ℃. The alkaline solution contains (BS, in mM) 2.5KCl, 152NaCl, 10 glucose, 2CaCl₂And 10HEPES, pH 7.4. Fluorescence was visualized using Ex/Im 494/5006 and a CCD camera (SensiCam) using a monochromatic light source (TILL Photonics GmbH). The chemicals were applied using a rapid infusion system (RSC-200, BioLogic Science). To quantify the difference in calcium transient amplitude, the ratio values were normalized by subtracting the baseline and further dividing by the calcium transient amplitude of ionomycin application for 2 seconds. Data were pre-analyzed offline using FEI offline analysis (TILL Photonics) and automatically further analyzed using MatLab. Unless otherwise indicated, quantitative data are presented as mean ± SEM. The number of experiments is indicated by n. For nonparametric data, significance was assessed using the ANOVA test or the Mann-Whitney t test. Statistics ofSignificant difference in was set as p<0.05 and x p<0.01。

Cell culture experiments

SV-40 immortalizes human microglial cell lines. For imaging experiments, SV-40 immortalized human microglia cell lines endogenously expressing piezo-1 and piezo-2R were used. The SV-40 cell line was maintained continuously. Cells were grown in culture T-25 flasks (Starstedt) initially coated for 1 hour with collagen 1 (rat tail, Gibco) in DMEM + GlutaMAX (Dulbecco's modified Eagle Medium, Gibco) supplemented with 10% FBS (Gibco) and 1% Str/Pen.

And (5) culturing trigeminal neurons. Trigeminal neuron cultures were prepared as described previously (Abushik PA et al 2017). Briefly, trigeminal ganglia were isolated from P10 Wistar rats and enzymatically dissociated in a solution of trypsin (0.25mg/mL, Sigma-Aldrich Co) and collagenase type I (760U/mL, Sigma-Aldrich Co) with continuous mixing (850rpm) at 37 ℃ for 15 minutes. Then, cells were plated on poly-L-lysine pre-coated coverslips (0.2mg/mL, Sigma-Aldrich Co) and cultured in F12 Nutmix + GlutaMAX medium (Gibco Invitrogen) supplemented with 10% fbs (Gibco Invitrogen) at 37 ℃ under 5% Co2 for 48 hours before measurement.

Apoptosis assay in N2a cells. N2a cells were treated with 5 μ M Yoda1 or vehicle for three consecutive days and exposed to 1% hypoxia for 24 hours as described above, and then cells were collected for apoptosis assay. In annexin V binding buffer (10 mM HEPES, 150mM NaCl, 2.5mM CaCl in PBS)₂pH 7.4), apoptotic cells were labeled with APC Annexin Ready Flow-dye (1:12.5 dilution, Invitrogen, Carlsbad, CA, USA). 4', 6-diamidino-2-phenylindole, dilactate (DAPI; Invitrogen, Carlsbad, Calif., USA) was added at a dilution of 1:3300 to stain to mark late apoptotic and necrotic cells. Samples were run using a CytoFLEX S instrument (Beckman Coulter Life Sciences, Indianapolis, IN, USA) and the results were analyzed using CytExpert software (version 2.3.0.84, Beckman Coulter Life Sciences, Indianapolis, IN, USA). The results of the hypoxic samples were normalized to the corresponding normoxic samples.

Measurement of primary cortical neuronal process growth. Mouse embryos from embryonic day 15Primary cortical neuron cultures were prepared from fetuses. The cortex was dissected and the tissue was dissociated with 0.0125% trypsin (15 min at 37 ℃, Sigma-Aldrich, st. louis, MO, USA). After trypsin inactivation and washing, cells were counted and plated at a density of 125000 cells/well on 48 well plates (coated with poly-D-lysine, Sigma-Aldrich, st.louis, MO, USA) or at a density of 180 ten thousand cells/cell on 6 well plates in Neurobasal medium supplemented with 2% B27 and 500 μ ML-glutamine (all from ThermoFisher Scientific, Waltham, MA, USA) and 10 μ g/ML gentamicin (Sigma Aldrich, st.louis, MO, USA). On

day

3 or 5 of in vitro culture (DIV), neurons were fed by changing 50% of the medium. Placing neurons in DIV 0

S3 live cell analysis system (Essen BioScience ltd., Hertfordshire, UK) and treated once with 5 μ M Yoda1(Tocris, Bio-Techne ltd., Abingdon, Oxfordshire, UK) in DIV 0, or once daily, until DIV 7, images were taken every 8 hours for analysis of neurite length and branch points.

Differentiation of microglia. Differentiation of microglia was performed as described previously but with minor modifications (Abud et al, 2017). Shortly, the iPSC was dissociated into single cells at D0 with 0.5mM EDTA or Accutase and replated onto matrigel at a density of 6-,000-16,000 cells/cm 2 in E8, 0.5% penicillin/streptomycin (P/S, 50IU/50mg/mL), 5ng/mL BMP4, 25ng/mL activin a (both from Peprotech or Miltenyi Biotec), 1. mu.M CHIR 99021(Axon or Stem Cell Technologies), and 10. mu. M Y-27632 to induce mesodermal differentiation. To increase differentiation efficiency, cells were treated at 5% O₂、5％CO₂And maintained under low oxygen conditions at 37 ℃. At D1, the medium was replaced with a lower concentration of 1. mu. M Y-27632. After 48 hours, at D2, the medium was changed to differentiation basal medium (dif-base) containing DMEM/F-12, 0.5% P/S, 1% GlutaMAXTM, 0.0543% sodium bicarbonate (all from Thermo Fisher Scientific),64mg/l L-ascorbic acid and 14. mu.g/l sodium selenite (all from Sigma). With 100ng/ml FGF2,50ng/ml VEGF (all fromPeprotech),10 μ M SB431542(Selleckchem or Stem Cell Technologies) and 5 μ g/ml insulin (Sigma) to supplement dif-base to induce the formation of hematopoietic endothelium. At D4, the medium was replaced with dif-base supplemented with 5. mu.g/ml insulin, 50ng/ml FGF2, VEGF, IL-6 and TPO, and 10ng/ml IL-3 and SCF to support the production and proliferation of EMP. From now on, the cells were kept in an normoxic incubator. Fresh EMP medium was changed daily until D8, at which time floating round EMPs were collected from the top of the monolayer. After centrifugation at 300x g for 5 minutes, 350,000 cells/ml were transferred to microglia medium in ULA dishes (Corning) containing IMDM (thermo Fisher scientific), 0.5% P/S and 10% heat-inactivated FBS (Biowest) or DMEM/F12, 0.5% N2, 0.5% B27 (supplemented with 5. mu.g/ml insulin, 5ng/ml MCSF and 100ng/ml IL-34 (both from Peprotech)). At D10, the cell suspension was altered by centrifugation and 350000 cells/ml were plated back into the ULA plates in microglia maturation medium supplemented with 10ng/ml MCSF and 10ng/ml IL-34. The medium was similarly changed every other day until D16, at which time the cells were detached from the ULA dishes with Accutase and replated at the required density on PDL-coated (Sigma) culture flasks (nunclon) cell culture-treated plates (Thermo Fisher Scientific) for the experiments. Half of the maturation medium was changed daily until the experiments were performed D23-D24.

Phagocytosis assay. To address whether activation of piezoelectric 1R affects phagocytosis, an important function of microglia, the inventors used hiPSC-differentiated microglia in a living cell analysis system (h

S3, Sartorius), which has a bio-particle with pH-sensitive conjugated probes (Thermo Fisher Science). Cells were plated in 96-well plates and pretreated 24 hours prior to assay with the following test compounds: vehicle (IMEM, Iscove modified Dulbecco medium, Gibco), with 0.5% P/S + IL-34(Peprotech) + MCSF (macrophage colony stimulating factor, Peprotech), LPS20ng/mL (e.coli (Escherichia Coli), serotype O111:b4, Sigma), 5 μ M Myoda1(Tocris), 50 μ M gadolinium chloride (Tocris), LPS + Yoda1(20ng/mL,5 μ M), LPS + gadolinium (20ng/mL,50 μ M) and LPS + Yoda1+ gadolinium (20ng/mL,5 μ M,50 μ M). At the same time as the pretreatment, pHRodo green zymosan bioparticles conjugate was added for phagocytosis (Thermo Fisher Scientific). Imaging was then performed every 15 minutes in a live cell assay system for 5 hours. In addition, fluorescence levels were measured and compared using Incucyte S3 software (Essen Bioscience).

And (4) measuring the cell factors. The effect of the piezoelectric 1R effect on the ability of iPSC-derived microglia to release cytokines was evaluated using a cytokine bead assay (CBA, PD Pharmingen). Shortly before the experiment, IPSC was administered at 70,000 cells/cm²Seeded in 24-well plates and cultured for 48 hours. Cells were cultured in differentiation medium and treated with 20ng/mL LPS or 5. mu.M Yoda1, 50. mu.M gadolinium chloride, LPS + Yoda1(20ng/mL, 5. mu.M), LPS + gadolinium (20ng/mL, 50. mu.M) and LPS + Yoda1+ gadolinium (20ng/mL, 5. mu.M, 50. mu.M). Next, the medium was collected, centrifuged to remove any cell debris, and the supernatant was frozen at-70 ℃. Cytokines were analyzed using the flow cytobead array mouse inflammation kit (BD Biosciences) according to the manufacturer's instructions.

And (4) carrying out cytotoxicity test. Cytotoxicity of in vitro cultures was assessed by Cytotox Green assay (Essen Bioscience) using the Incucyte S3 live cell assay system (Essen Bioscience, Ann Arbor, MI, USA, # 4647). Microglia were assayed at a density of 15000 cells/well in poly-D-lysine (PDL, Sigma, # P0899) coated culture flasks (Thermo Scientific, #167008) or ImageLock 96 well plates (Essen Bioscience, # 4379). On the day of assay, cells were treated with 0.1-20 μ MYoda1(Tocris, #5586) in PM medium supplemented with 250nM Cytotox Green reagent (Essen Bioscience, # 4633). Equal volumes of DMSO (Sigma, # D2650) were used as vehicle and 200 μ M MPP + (Sigma, # D048) as positive control. At 37 ℃ and 5% CO₂Next, cells were imaged in vivo in IncuCyte S3 every 3 hours for 3 days to obtain cell death over time. Capture 1 to 2 10x images per well, using phase contrast mode to obtain fusion and green fluorescence mode to obtainThe fluorescence intensity was obtained. Quantification was performed using Inocyte S3 software (2019B). Green fluorescence was separated from the background by top-hat thresholding (top-hat thresholding) and all settings were kept constant between different conditions. As a result measurement, the green integrated intensity (GCU) of each image was divided by the fused area of each image at each time point and the data was normalized to the maximum of the positive control or vehicle to combine the average group values from multiple experiments in the same plot.

Animal experiments

All animal work was approved by the animal care and use committee of the University of Eastern Finland (Kuopio) and conducted according to the animal care guidelines of the national institutes of health.

To evaluate the effect of microglia piezoelectric 1R mechanical sensors in vivo, the inventors used 5-month-old transgenic 5XFAD male mice (Jackson Laboratories, Bar Harbor, Maine, US) randomly divided into 3 groups. The 5xFAD mice express the human APP and PSEN1 transgenes, which have 5 AD-associated mutations: swedish (K670N/M671L), Florida (I716V) and London (V717I) mutations in APP, and M146L and L286V mutations in presenilin-1 (PSEN 1). These widely used mice recapitulate many AD-associated phenotypes and have relatively early and aggressive manifestations. Amyloid plaques, with gliosis, are found in mice as small as two months of age. Female amyloidosis is more severe than male. Neuronal loss occurs in multiple brain regions, beginning at approximately 5 months in the most prominent areas of amyloidosis. Mice exhibit a range of cognitive and motor deficits. Animals were implanted with a ventricular cannula. Briefly, surgical anesthesia was induced using 5% isoflurane and 1.8% isoflurane (at 30% O)₂/70％N₂O in). The temperature of the animals was maintained at 37. + -. 0.5 ℃ using a thermostatically controlled heating blanket (PanLab, Harvard Apparatus) with a rectal probe. After exposing the skull, a small hole of about 1mm in diameter was drilled in the left hemisphere of the skull using the following coordinates: m/l (medial/lateral)) +1.1mm, a/p (anterior/posterior) -0.3mm, d/v (dorsal/ventral) -2.0 mm. Then specially designed chronicThe cannula (cannula infusion system; Plastic1, preclinical study assembly) was mounted throughout the bore and fixed to the mouse head (using a dental mount). After implantation, animals were placed in individual cages for 48 hours of recovery, followed by infusion of Yoda1(0.05 μ g/μ l), GdCl (0.15 μ g/μ l), or saline + 1% DMSO by cannula, once daily for the next 2 weeks, with 2 days of rest and 5 days per injection (10 days total). Mice were infused with 5 μ l of pre-retained (stemmed) drug. Next, mice were euthanized 6h after the last infusion to collect tissues. Mice were anesthetized with an excess of Avermectin and then perfused cardiovertially with heparinized saline (2500 IU/L). The ICV-infused hemispheres were removed and post-fixed in 4% PFA and then cryoprotected in 30% sucrose.

Brain sample preparation and immunohistochemistry. The left hemisphere of the brain (injected) was frozen in liquid nitrogen, cryosectioned into 20- μm sagittal sections, and stored in cryoprotective solution. Six consecutive sagittal brain sections at 400 μm intervals were selected from each mouse for immunohistological staining. Then, a β deposits and microglia were detected. The inventors used a primary antibody specific for human A β 4-10 amino acids (WO2, Sigma Millipore, dilution 1: 1000) and an antibody specific for Iba-1 protein (Iba1, Wako, dilution 1: 250) overnight at RT and visualized further by fluorescent Alexa 568 and Alexa 488 secondary antibodies (dilution 1:500, ThermoFisher Scientific) accordingly. To quantify the a β and Iba1 immunoreactivity, stained sections were imaged at 10-fold magnification under a Zeiss Axio imager m.2 microscope equipped with an Axiocam 506mono CCD camera (Carl Zeiss, Oberkochen), running ZEN software (Carl Zeiss) for smearing and stitching of the images. Cortical and hippocampal a β and Iba1 immunoreactions were quantified from 6 stained sections at 400 μm intervals per animal using MatLab code (MathWorks, MatLab 2017 b). The accuracy of the analysis was confirmed by re-analyzing the partial images using ImageJ 1.50i software. Data are presented as mean ± SEM.

Permanent middle cerebral artery occlusion to mimic ischemic stroke and treatment with Yoda. Balb-c mice received permanent middle cerebral artery occlusion (pMCAo). Anesthesia was induced with 5% isoflurane in 30% oxygen and 70% nitrogen as a carrier gas and maintained with 2% isoflurane during surgery. The temperature was kept constant (37. + -. 1 ℃) using a heating blanket (Harvard appaatus, PanLab, Barcelona, Spain) attached to a rectal probe. The temporal muscle was separated from the skull and a hole (1 mm diameter) was drilled in the temporal bone. After removal of the dura mater, the exposed left MCA was lifted and blocked using a thermal regulator (Aaron Medical Industries inc., Clearwater, FL, USA). The success of the occlusion was confirmed by cutting the artery, after which the temporal muscle was placed back on top of the hole and the skin was sutured closed. The mice were returned to their cages and recovered from surgery. Sham operated animals were treated similarly except for MCA occlusion.

Yoda1(Tocris, Bio-Techne Ltd., Abingdon, Oxfordshire, UK; 1.78mg/kg) was delivered intravenously (i.v.) at a frequency similar to that of HX600 administration in study I. A50 mM stock of Yoda1 was first prepared in dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, Mo., USA) and then diluted with 0.9% sterile saline (Baxter, Deerfield, IL, USA) to obtain a solution of the desired concentration. Vehicle solutions were prepared by diluting the corresponding amount of DMSO into saline. In addition, other solvents than DMSO may also be used.

Adhesive removal test for evaluation of sensorimotor performance. pMCAo-induced sensorimotor deficits were tested using the adhesive removal test as described previously (Loppi et al, 2017). For the adhesive removal test, each mouse was given four training sessions before ischemia was induced. In the first training, mice were habituated to the test environment without tape, and in the second and third training, mice were placed in test chambers with adhesive. The fourth session was performed the day before ischemia was induced and was used to record the baseline time for the perception and removal batch (batch). Actual tests were performed at 1dpi and 3dpi prior to MRI.

And (5) measuring the size of the focus. Lesion volumes were measured in vivo by Magnetic Resonance Imaging (MRI) using a vertical 9.4T Oxford NMR 400 magnet (Oxford Instrument PLC, Abington, UK) at 24 and 72 hours post-ischemia. Mice were anesthetized with 5% isoflurane and maintained with 1% isoflurane during imagingAnd (6) anaesthetizing. Acquisition of a multi-slice T2 weighted image (repetition time 3000ms, echo time 40ms, matrix size 128 × 256, field of view 19.2 × 19.2mm²Slice thickness 0.8mm and number of slices 12) and the images obtained were analyzed in Matlab environment (Math-works, nature, MA, USA) using the in-house made Aedes software.

Red Blood Cell (RBC) Ca2+ flux

Blood samples (50-100 μ l) were collected from 5xFAD mice of different ages (13-15 weeks, 15-17 weeks, 17-19 weeks, 19-21 weeks, and 21-23 weeks) for analysis of RBC Ca2+ flux. Soon, blood samples were collected in Eppendorf tubes containing sodium citrate to prevent clotting. The sample was then filtered through a 100 μm filter to remove possible contaminants and aggregates, followed by two washes with PBS (5 min, 300g, RT) to remove plasma. 50 μ l of red blood cells were transferred to a clean tube and resuspended in 200 μ l PBS (total volume of 250 μ l cell suspension). Mu.l Fluo4 stock solution was added and the cells were incubated for 30 min at 37 ℃ in the dark. The cell suspension was then washed twice with PBS followed by one wash with HBSS (5 min, 300g, RT). The supernatant was carefully removed by pipette and 50. mu.l of the pellet was resuspended in 450. mu.l of HBSS to form a 10% cell suspension. Mu.l of the 10% cell suspension was transferred to 410. mu.l of RPMI/FBS (10: 1). At a high flow rate of 60. mu.l/min, Ca2+ flux was recorded by CytoFlex. Recording was started 20 seconds after the flow rate stabilized. After a 20 second recording (baseline fluorescence), Yoda1 was applied at a final concentration of 10 μ M, ionomycin was added at a final concentration of 10 μ M at the 4 minute 20 second time point (4 minutes after addition of Yoda 1) as a positive control, and the sample was run on for up to 6 minutes. Mean fluorescence intensity was measured using gating at different time points-baseline (before Yoda 110 μ M administration), recorded 1 min, 2 min, 3 min and 4 min.

Example 1

piezoelectrics

1 and 2 are mechanosensory channels in microglia cell types

In the present invention, piezoelectric receptors are found in human induced pluripotent stem cell (hlsc) -derived microglia. This clinically highly relevant model recapitulates the phenotype of microglia at the developmental, functional and transcriptome levels and expression of microglia markers, giving us an unprecedented opportunity to study microglia in the human environment.

The qRT-PCR data revealed the expression of mechanosensitive piezoelectric 1R in all types of microglia tested. To fully quantify the presence of piezoelectric receptor expression levels, the inventors normalized their data to piezoelectric receptor expression in mouse trigeminal neurons (mTG, fig. 1A, fig. 1B) known to contain two types of receptors. The present inventors found that mouse astrocytes exhibit both high-level expression of the piezomechanosensitive channel (piezo 1:0,8474 ± 0,2551; piezo 2:0,8576 ± 0,2042, n ═ 4, fig. 1A, fig. 1B). Between the microglia types tested only the cell line SV-40 (piezo 1:0,1188 ± 0,0458; piezo 2:0,9901 ± 0,5015; n ═ 4) and mouse microglia (piezo 1: 0.0114 ± 0.0012; piezo 2: 0.0019 ± 0.0008; n ═ 4) demonstrated the presence of two mechanosensitive receptors. However, current data indicate that human postmortem microglia, human iPSC-derived microglia, and BV2 (immortalized mouse microglia) cell lines express only piezoelectric 1R (hpM:0.0018 ± 0.0008, n ═ 4; hipsccm: 0.000434 ± 0.000003, n ═ 4; BV2:0,0229 ± 0,0031, n ═ 4). Consistent with the qPCR data, the inventors demonstrated piezoelectric 1R morphological expression by IHC in hipsc-derived microglia as shown in fig. 1C and 1D.

Example 2 Yoda-1 activation of piezoelectric 1Ca in human microglia²⁺Influx and reversal of the Abeta Effect

Use of Ca in hipSC cells and SV-40 cell lines following the first discovery of mechanosensory in microglia²⁺Imaging tests the functional effect of piezo 1R in microglia. The hiPSC-derived microglia was selected to be closest to the human postmortem microglia level by the expression level of the piezoelectric 1R cell line, and the SV-40 cell line was selected as the cell line having the highest expression level. In addition to mechanical stimulation, the piezoelectric 1R (but not piezoelectric 2) channels can be co-activated with small molecules called Yoda 1. The unique opportunity is utilized to activate the natural piezoelectric 1R mechanical sensitive typeThe inventors used 50 μ M Yoda1 diluted in the base solution, applying a fast solenoid valve based perfusion system (Biologic 2000) that additionally generated a small hydraulic pressure at the beginning of the application. The results show that, although the expression of piezo 1R was low in microglia compared to TG neurons and astrocytes, these cells expressed enough native piezo 1R on the membrane to activate strong Ca-influx in the two tested microglia lines (fig. 1E top trace).

Further evaluated whether low concentrations of soluble A β in the early AD model would affect native piezoelectric 1-activated Ca in human microglia²⁺And (4) internal flow. Based on preliminary tests, Ca was activated even at low A.beta.concentrations²⁺Influx, therefore a short (15 min) pre-incubation protocol was employed to avoid overlapping with the piezoelectric 1R stimulation activated by Yoda 1. Control hiPSC microglia were preincubated in BS for the same time. Control cells showed strong Ca induced by short (2s) application of Yoda1²⁺Transient (1.15 ± 0.36, n ═ 6; fig. 1E). Microglia-like cells preincubated with 20pM soluble Α β (diluted in BS) strongly inhibited piezoelectric 1RCa activated by Yoda1²⁺(iv) inflow (0.27 ± 0.08, p ═ 0.0073, n ═ 11; fig. 3E, fig. 3F). Next, a pre-incubation in the non-specific piezo1 inhibitor GdCl (50. mu.M in BS) was performed, which adds Ca²⁺Transients were suppressed by 90% (0.10 ± 0.1, p ═ 0.0073, n ═ 5; fig. 1E, fig. 1G).

The SV-40 cell line (which the present inventors found to express the highest piezoelectric 1R levels and even higher piezoelectric 2R levels than other microglia) was then Ca-plated²⁺A similar pre-incubation protocol was used in the imaging experiments. The data demonstrate A β preincubation on Ca of SV-40 cell lines ²⁺50% inhibition of transients (0.82 ± 0.14, n ═ 13 vs. 0.44 ± 0.05, n ═ 14; p ═ 0.0085; fig. 1H), which can be explained by piezoelectric 2 mechanically induced (by hydraulic actuation of rapid perfusion systems) Ca2+ influx, possibly with uninhibited fraction of a β, because of the SV-40 cell line's Ca²⁺The input expresses two types of piezoelectric channels. This hypothesis yielded Ca activated by preincubation of GdCl for Yoda1 in SV-40 cells²⁺Strong suppression of transients (0.82 ± 0.14, 13 for n + 13 versus 0.17 ± 0.04, 13 for n; p ═ 0.00009) was supported. To further test the hypothesis, the inventors tested Ca induced by BS application alone on the control SV-40 using their rapid perfusion system²⁺The range of transients.

This data indicates that low concentrations of soluble a β strongly inhibit native piezoelectric 1R in human microglia, which can lead to their passage of inhibited Ca²⁺Signal transduction is sensitive to mechanical stimuli in the brain.

Example 3 activation of Yoda1 reduces the proinflammatory Properties of microglia

Piezoelectric activation and inhibition have functional consequences in microglia, since specific activation of piezoelectrics by Yoda1 significantly reduced LPS-induced microglia pro-inflammatory activation (increased pro-inflammatory cytokine secretion), suggesting that piezoelectric activation beneficially modulates microglia function (fig. 2A). Furthermore, Yoda1 activation of Piezo significantly protected iPSC-microglia from cytotoxicity (fig. 2B)

Example 4 Yoda1 enhances microglial phagocytosis in vivo.

To provide proof-of-concept of the effect of Yoda1 on enhancing microglial phagocytosis, 5xFAD transgenic mice mimicking alzheimer's disease were treated with Yoda1 or the piezoelectric inhibitor gadolinium (GdCl) for 2 weeks. Brain a β burden and microglial proliferation of the brain were analyzed by immunohistochemical staining. The results show that Yoda1 treated mice showed significantly enhanced Iba1 immunoreactivity (fig. 3A), significantly reduced brain a β burden in cortex and hippocampus (fig. 3B), and enhanced microglial accumulation near a β deposits (fig. 3C).

Example 5. primary neurons express piezoelectricity during their development.

To demonstrate that piezo-electric 1 is expressed in cerebral microglia, immunohistochemical staining of piezo-electric was performed. Surprisingly, piezoelectricity is also expressed in neurons, also in the hippocampal dentate gyrus where neurogenesis is known to occur. This allows the inventors to test the effect of Yoda1 on specifically inducing piezoelectric 1 activation in neurons. The data show that primary neurons express piezoelectricity during their development (fig. 4A). Thus, when primary neurons were treated with Yoda1, the inventors were able to show that Yoda1 treated neurons produced significantly wider branches (fig. 4B and 4C) and neurite length (fig. 4D) than their vehicle-treated control cells. This means that Yoda1 is able to promote neuronal development.

Example 6 Yoda1 also exerts a protective effect in the case of ischemic stroke.

Since modulation of microglia function and enhancement of neurogenesis are considered as potential therapeutic approaches, Yoda1 is also presumed to play a protective role in the case of ischemic stroke. The inventors first analyzed whether Yoda1 could prevent hypoxia-induced apoptosis and could demonstrate that Yoda1 is highly effective in preventing hypoxia-induced early and late apoptosis in vitro (fig. 5A, 5B). To assess whether Yoda1 has a protective effect on ischemia-induced sensorimotor defects, balbc-mice were subjected to permanent middle cerebral artery occlusion and analyzed for the ability of Yoda1 treatment to prevent ischemia-induced defects in the cement removal test. In fact, in this sensorimotor test, mice treated with Yoda1 were comparable to sham operated controls. Yoda1 was administered immediately after the stroke, once daily for three consecutive days thereafter. Lesion size was measured on MRI at 1DPI and 3 DPI. Yoda1 treated mice showed significantly smaller lesion sizes at both time points (fig. 5C). Representative MRI images (fig. 5D) from vehicle (upper panel) and Yoda1 treated mice (lower panel), respectively. Yoda1 treated ischemic mice had no apparent deficit in their ability to feel sticky plaques (patch) after stroke (fig. 5E). All data are expressed as mean +/-SEM, where p 05, p 01, p 001, as analyzed by t-test or two-way ANOVA, followed by Bonferroni post-hoc tests.

Example 7 piezoelectric receptor activation by Yoda1 as a functional biomarker for Alzheimer's disease.

Since the piezoelectric receptor can be blocked by α β -peptide, the present invention assumes that its functionality is reduced under AD conditions. In fact, the inventors demonstrated that Yoda1 elicited significantly lower responses in the blood of Transgenic (TG)5xFAD mice compared to their age-matched Wild Type (WT) controls (fig. 6A-6E). Blood samples were drawn from AD transgenic mice (TG) of different ages and their wild type controls (WT) for analysis of red blood cell calcium flux induced on yoda 1. Cells were stimulated with 10uM of the piezoelectric agonist Yoda1 and their calcium response was performed on the cytoflex. TG mouse blood cells reacted at lower intensities compared to age-matched WT controls at 17-23 weeks of age, suggesting that Yoda response may serve as a functional biomarker for AD.

The main result is that murine piezoelectric channels have reduced their activity in the early stages of AD pathology due to a reduction in calcium flux in RBCs activated by the piezoelectric 1 agonist Yoda 1. The method comprises the following steps: 1) washing a small volume (100 μ l) of blood sample containing citrate to prevent clotting with PBS, resuspending the RBC pellet in PBS after centrifugation; 2) loading the sample with calcium indicator Fluo4 AM; 3) after loading, the cells were washed, the supernatant removed, and the pellet resuspended in HBSS to give a 10% cell suspension; 4) transferring the sample to RPMI/FBS; 5) samples were used to record intracellular calcium dynamics in a time-lapse mode using the CytoFlex machine. 6) Mean fluorescence intensities were measured at baseline and at various time points (after application of Yoda 1). This data suggests that piezoelectric receptor activation is dysfunctional in AD and can be used as a functional biomarker for AD.

Example 8.Ca²⁺Flow method.

Blood samples (50-100. mu.l) were collected in Eppendorf tubes containing sodium citrate to prevent clotting. The sample was then filtered through a 100 μm filter to remove possible contaminants and aggregates, followed by two washes with PBS (5 min, 300g, RT) to remove plasma.

After centrifugation of the sample solution, the red blood cells form a pellet. 50 μ l of red blood cells were transferred to a clean tube and resuspended in 200 μ l PBS (total volume of 250 μ l cell suspension). Mu.l Fluo4 stock solution was added and the cells were incubated for 30 min at 37 ℃ in the dark.

The cell suspension was washed twice with PBS and then once with HBSS (5 min, 300g, RT). The supernatant was carefully removed with a pipette and 50. mu.l of the pellet was resuspended in 450. mu.l of HBSS to form a 10% cell suspension. Mu.l of the 10% cell suspension was transferred to 410. mu.l of RPMI/FBS (10: 1). The final volume was 420. mu.l, which was sufficient for CytoFlex to record for a maximum of 7 minutes at a high flow rate of 60. mu.l/min.

The sample was recorded for 6 minutes. Recording was started 20 seconds after the flow rate stabilized. After 20 seconds recording (baseline fluorescence), Yoda1 was applied at a final concentration of 10 μ M, ionomycin was added at a final concentration of 10 μ M as a positive control at the 4 minute 20 second time point (4 minutes after addition of Yoda 1), and the sample was allowed to run for up to 6 minutes. Mean fluorescence intensity was measured using gating at different time points-baseline (before Yoda 110 μ M administration), recorded 1 min, 2 min, 3 min and 4 min.

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Claims

1. A piezoelectric agonist for use in the treatment of a neurodegenerative and/or neuroinflammatory disease, or a condition or disorder associated with a neurodegenerative and/or neuroinflammatory disease.

2. The piezoelectric agonist for use in therapy according to claim 1, wherein the piezoelectric is piezoelectric 1 or piezoelectric 2.

3. The piezoelectric agonist for use in therapy according to claim 1 or 2, wherein the piezoelectric is from a mouse or a human.

4. The piezoelectric agonist for use in therapy according to any one of claims 1-3, wherein the piezoelectric agonist is for modulating microglial cell function.

5. The piezoelectric agonist for use in therapy according to claim 4, wherein the microglial cell function to be modulated is motility, phagocytosis and/or cytokine release.

6. A piezoelectric agonist for use in therapy according to any one of claims 1 to 5, wherein the agonist is for use in activating piezoelectricity.

7. The piezoelectric agonist for use in therapy according to any one of claims 1-6, wherein the agonist is selected from the group consisting of: yoda1, Jedi1, Jedi2, and functional analogs thereof.

8. The piezoelectric agonist for use in therapy according to any one of claims 1-7, wherein the agonist is Yoda 1.

9. The piezoelectric agonist for use in therapy according to any one of claims 1-8, wherein amyloid beta accumulation is inhibited.

10. The piezoelectric agonist for use in treatment according to any one of claims 1 to 8, wherein the load of amyloid β plaques is reduced.

11. The piezoelectric agonist for use in therapy according to any one of claims 1 to 10, wherein the piezoelectric agonist is used in combination with at least one molecule selected from the group consisting of: arachidonoyl ethanolamide, 2-arachidonoyl glycerol, palmitoyl ethanolamide, oleoyl ethanolamide, and linoleoyl ethanolamide.

12. The piezoelectric agonist for use in treatment according to any one of claims 1 to 11, wherein the neurodegenerative and/or neuroinflammatory disease, or a condition or disorder associated with the neurodegenerative and/or neuroinflammatory disease is selected from the group consisting of: alzheimer's disease, stroke, Parkinson's disease, head trauma, cerebral amyloid angiopathy, spongiform encephalopathy, cerebral amyloid diseases and pruritus.

13. A piezoelectric agonist for use in a pharmaceutical composition for prophylactic or therapeutic treatment of a neurodegenerative and/or neuroinflammatory disease, or a disorder or condition associated with a neurodegenerative and/or neuroinflammatory disease, for treatment according to any one of claims 1-12.

14. A method of treating a neurodegenerative and/or neuroinflammatory disease, or a disorder or condition associated with a neurodegenerative and/or neuroinflammatory disease, the method comprising administering to a subject in need thereof a piezoelectric agonist, wherein the agonist is for activating piezoelectric.

15. The method of claim 14, wherein the piezoelectric agonist is selected from the group consisting of: yoda1, Jedi1, Jedi2, and functional analogs thereof.

16. The method of claim 15, wherein the piezoelectric agonist is Yoda 1.

17. The method of any one of claims 14-16, wherein the piezoelectric agonist is used in combination with at least one molecule selected from the group consisting of: arachidonoyl ethanolamide, 2-arachidonoyl glycerol, palmitoyl ethanolamide, oleoyl ethanolamide, and linoleoyl ethanolamide.

18. The method of any one of claims 14-17, wherein the condition, disease or disorder is or is associated with at least one neurodegenerative and/or neuroinflammatory disease selected from the group consisting of: alzheimer's disease, Parkinson's disease, stroke, head trauma, cerebral amyloid angiopathy, spongiform encephalopathy, cerebral amyloid diseases and pruritus.

19. A method for determining the risk associated with the development or presence of a neurodegenerative and/or neuroinflammatory disease, or a condition or disorder associated with said neurodegenerative and/or neuroinflammatory disease, in a human subject, comprising the steps of:

a. providing a test sample and a control sample;

b. measuring the baseline fluorescence intensity of the test sample and the control sample;

c. adding a piezoelectric agonist to the test sample;

e. determining the difference in fluorescence intensity of the test sample and the control sample, wherein a decrease in fluorescence intensity of the test sample compared to the fluorescence intensity of the control sample in step e is indicative of a decrease in piezoelectric receptor activity in the test sample, and is indicative of the risk of development or presence of a neurodegenerative disease and/or neuroinflammatory disease or a disorder or condition associated with the neurodegenerative disease and/or neuroinflammatory disease in the human subject.

20. The method of claim 19, wherein the test sample and control sample consist of red blood cells, serum, plasma, or whole blood or blood mononuclear cells.

21. The method of claim 20, wherein the test sample and control sample consist of red blood cells or blood mononuclear cells.

22. The method of any one of claims 19-21, wherein the neurodegenerative and/or neuroinflammatory disease, or the disorder or condition associated with the neurodegenerative and/or neuroinflammatory disease, comprises at least one of the following diseases: alzheimer's disease, Parkinson's disease, stroke, head trauma, cerebral amyloid angiopathy, spongiform encephalopathy, cerebral amyloid diseases and pruritus.

23. A method of modulating microglial function in a microglia cell comprising contacting the cell with the piezoelectric agonist of any one of claims 1-13.